The central goal of this proposal is to understand molecular mechanisms of probiosis for new treatment and prevention strategies in inflammatory bowel disease. This proposal includes comprehensive genetic and peptidomic approaches in order to determine the molecular basis of anti-inflammatory functions by probiotics. Our laboratory has obtained evidence for novel mechanisms of probiosis including inhibition of pro-inflammatory TNFa production by macrophages. Intestinal lactobacilli have been isolated and tested for tumor necrosis factor-alpha (TNF-a)-inhibitory activity. The overall hypothesis is that probiotic intestinal Lactobacillus strains secrete peptides that suppress TNF-a signaling in macrophages and inhibit intestinal inflammation. The functions of candidate Lactobacillus genes that regulate probiotic activities including peptide secretion will be studied in vitro using macrophages activated by Toll-like receptor agonists. Wild type lactobacilli and knockout Lactobacillus strains lacking anti-inflammatory effects in vitro will be administered to IL-10-deficient mice in order to explore mechanisms of probiotic action in vivo. Identification of probiotic genes encoding anti-inflammatory functions and probiotic response pathways in macrophages may provide valuable insights into potential human targets for probiotic action in Crohn's disease. 1. Specify probiotic anti-inflammatory genes in intestinal Lactobacillus strains by knockout mutagenesis. 2. Identify anti-inflammatory glutamate-containing peptides by comparative peptidomics of the Lactobacillus secretome. 3. Compare mucosal inflammatory responses in the intestines of IL-10-deficient mice colonized with knockout or wild type isogenic strains of probiotic Lactobacillus. These studies will facilitate the identification of beneficial bacteria that can be used to treat inflammatory bowel disease such as Crohn's disease and ulcerative colitis. We seek to understand the mechanisms of action of beneficial intestinal bacteria so that new strains can be identified naturally or engineered for human medicine.